WO2002080934A1

WO2002080934A1 - A composition comprising glucosamine for treating angiogenesis-dependent diseases

Info

Publication number: WO2002080934A1
Application number: PCT/KR2002/000622
Authority: WO
Inventors: Min Young Kim; Byung Young Park; Chang Hee Moon; Eun Kyu Park
Original assignee: Angiolab Inc.
Priority date: 2001-04-09
Filing date: 2002-04-09
Publication date: 2002-10-17
Also published as: KR20020078328A; KR100465229B1

Abstract

The present invention shows that 2-amino-2-deoxy-D-glucopyranose inhibited angiogenesis. Therefore, a composition is provided for treating or preventing angiogenesis-dependent diseases, which comprises a therapeutically effective amount of 2-amino-2-deoxy-D-glucopyranose or salt thereof.

Description

A COMPOSITION COMPRISING GLUCOSAMINE FOR TREATING ANGIOGENESIS-DEPENDENT DISEASES

Field of the Invention

The present invention relates to a composition for treating or preventing angiogenesis-dependent diseases, which comprises a therapeutically effective amount of 2-amino-2-deoxy-D-glucopyranose or a salt thereof.

Background of the Invention

2-Amino-2-deoxy-D-glucopyranose (often called simply as glucosamine) is an endogenous amino-monosaccharide. Its role in the body is to maintain and protect cartilage by activating the production of glycosammoglycan and proteoglycan in chondrocytes. Therefore, it can prevent recessive cartilage diseases without severe adverse effects. It is also known that glucosamine has anti-inflammatory activity due to its anti-protease activity.

Angiogenesis is the process of generating new capillary blood vessels, and it results from activated proliferation of pre-existing endothelial cells. Neovascularization is tightly regulated, and occurs only during embryonic development, tissue remodeling, wound healing and periodic cycle of corpus luteum development (Folkman and Cotran, Relation of vascular proliferation to tumor growth, Int. Rev. Exp. Pathol, 16, 207-248(1976)). The endothelial cells grow much more slowly than other types of cells in the body. However, if the proliferation rate of these cells is enhanced by the failure of regulation of angiogenesis, some pathological angiogenesis are induced (Timar, J. Pathol. Oncol. Res., 6, 85-94(2001)). Pathological angiogenesis is involved in many diseases. For example, cardiovascular diseases such as angioma, angiofibroma, vascular deformity, atherosclerosis, synechia and edemic sclerosis; and opthalmological diseases such as neovascularization after cornea implantation, neovascular glaucoma, diabetic retinopathy, angiogenic corneal disease, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, and granular conjunctivitis are related to angiogenesis. Chronic inflammatory diseases such as arthritis; dermatological diseases such as psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis and acne are also angiogenesis-dependent. Angiogenesis is also essential to metastasis and growth of cancer (DAmato RJ and Adamis AP, Ophthalmol, 102, 1261-1262(1995); Arbiser JL, J. Am. Acad. Derm., 34, 486-497(1996); O'Brien K.D., et al. Circulation, 93, 672-682(1996); Hanahan D and Folkman, J. Cell, 86, 353-364(1996)). New blood vessels provide not only nutrients and oxygen to fast-growing cancer cells, but also ways for metastasis to occur through the blood stream (Polverini P.J., Critical Reviews in Oral Biology, 6, 230-247(1995)). Currently, a large variety of chemo- and immunotherapies are applied in the treatment of cancer, but the efficacy of the therapies is limited due to the lack of anti-metastasis effects.

It has been reported that arthritis, a well-known inflammatory disease, is an autoimmune disease. The growth of vascular endothelial cells in the synovial cavity is activated by the inflammatory cytokines, finally destroying cartilage in the articulation (Kocb AE, Polverini PJ and Lcibovich SJ, Arth Rheum, 29, 471 -479(1986); Stupack DG, Storgard CM and Cheresh DA, Braz. J. Med. Biol. Res., 32, 578-581(1999); Koch AE, Arthritis Rheum, 41, 951- 962(1998)).

Many people lose eyesight because of various ocular diseases, e.g., the infiltration of capillary blood cells into the vitreous humor (Jeffrey MI and Takayuki A, J. Clin. Invest. , 103, 1231-1236(1999)).

Psoriasis is caused by uncontrolled proliferation of skin cells. Fast- growing cell requires sufficient blood supply, and abnormal angiogenesis is induced in psoriasis (Folkman J., J. Invest. Dermatol, 59, 40-48(1972)).

In order to prevent and/or treat the aforementioned angiogenesis-related diseases, it is required to develop an effective inhibitor of angiogenesis.

Summary of the Invention

Accordingly, it is a primary object of the present invention to provide a composition for treating or preventing angiogenesis-dependent diseases.

It is another object of the present invention to provide a method for treating or preventing angiogenesis-dependent diseases.

In accordance with one aspect of the present invention, there is provided a composition for treating or preventing angiogenesis-dependent diseases, which comprises a therapeutically effective amount of 2-amino-2- deoxy-D-glucopyranose or a salt thereof.

In accordance with another aspect of the present invention, there is provided a method for treating or preventing angiogenesis-dependent diseases by administering 2-amino-2-deoxy-D-glucopyranose or a salt thereof to subjects in need thereof in an amount effective for treating or preventing the diseases.

Brief Description of the Drawings

The above and other objects and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings, wherein:

Figs. 1A to ID respectively show effects of various forms of 2-amino-

2-deoxy-D-glucopyranose on the tube formation by human umbilical vein endothelial cells (HUVEC) grown on Matrigel (A: Control HUVEC; B: HUVEC treated with 1 mg/ml of glucosamine hydrochloride; C: HUVEC treated with 1 mg/ml of glucosamine-2-sulfate; and D: HUVEC treated with 1 mg/ml of glucosamine-6-phosphate);

Figs. 2A to 2C illustrate dose-dependent effects of 2-amino-2-deoxy-D- glucopyranose on HUVEC tube formation (A: Control HUVEC; B: HUVEC treated with 2.5 mg/ml of gluosamine-2-sulfate; and C: HUVEC treated with

0.25 mg/ml of gluosamine-2-sulfate);

Figs. 3 A and 3B show the anti-angiogenic effects of 2-amino-2-deoxy-

D-glucopyranose in chorioallantoic membrane assays (A: control; and B: glucosamine-2-sufate); and Fig. 4 demonstrates the effect of glucosamine hydrochloride on angiogenesis in mouse Matrigel model.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

2-Amino-2-deoxy-D-glucopyranose (glucosamine) can be extracted from the cartilage of various animals, synthesized by a conventional method, or purchased from a commercial source.

A pharmaceutically acceptable salt of glucosamine can be prepared with any pharmaceutically acceptable acid such as hydrochloric acid, bromidic acid, sulfuric acid, acetic acid, methansulfonic acid, propionic acid, glutaric acid, fumaric acid, maleic acid, tartaric acid, glutamic acid, glucuronic acid, galactronic acid, ascorbic acid, phosphoric acid, nitric acid, L-aspartic acid, lactic acid, vanilic acid and hydroironic acid. The glucosamine used in the present invention includes glucosamine hydrochloride, glucosamine 2-sulfate, glucosamine pentaacetate and the modified glucosamine such as glucosamine 6-phosphate, glucosamine 3-sulfate, glucosamine 6-sulfate, glucosamine 2,3- disulfate, glucosamine 2,6-disulfate, glucosamine 3,6-disulfate, glucosamine 1- phosphate and glucosamine 6-phosphate.

The anti-angiogenic effect of glucosamine or the salt thereof is confirmed not only in tube formation assay, but also in CAM assay and mouse Matrigel model. The tube formation assay is an in vitro experimental method, which is closely related to in vivo efficacy, and this assay is to investigate the effect of a candidate medicine on the migration and differentiation of human endothelial cells to form microvascular network. The in vivo angiogenesis can be investigated in CAM from fertilized eggs and mouse Matrigel model which enables to measure angiogenesis quantitatively.

It is therefore clear that the pharmaceutical composition of the present invention, which comprises glucosamine as an active ingredient in combination with pharmaceutically acceptable excipients, carriers or diluents, may be useful for the treatment of angiogenesis-related diseases. Examples of angiogenesis-dependent diseases include cancer, metastasis, angioma, angiofibroma, diabetic retinopathy, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis, acne and arthritis.

A pharmaceutical formulation may be prepared in accordance with any one of the conventional procedures.

In preparing the inventive composition, the active ingredient is preferably admixed or diluted with a carrier, or enclosed within a carrier, which may be in the form of a capsule, sachet or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient. Thus, the compositions may be in the form of tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, maltodextrin, saline, buffered saline, glycerol, ethanol, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propyl- hydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.

The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the well-known procedures in the art.

The composition of the present invention can be administered via a variety of routes including oral, intravenous, intraperitoneal, subcutaneous, intramuscular, intra-arterial, transdermal, rectal, nasal, ocular, and topical introduction. A typical daily dose of the active ingredient may range from about 50 to 2000 mg , preferably 100 to 1500 mg, and can be administered in a single dose or in divided doses. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the disease to be treated, the severity of the patient's symptom, the chosen route of administration, and the age, sex, and body weight of the individual patient, food, dosing time; and, therefore, the above dose should not be intended to limit the scope of the invention in any way. Further, other commercially available drugs can be co-administered with glucosamine of present invention to potentiate the effect of the composition. Moreover, glucosamine can be incorporated in foods or beverages, as an additive or a dietary supplement, for the purpose of preventing the angiogenesis-related diseases.

The following Examples are intended to further illustrate the present invention without limiting its scope; and the experimental methods used in the Examples can be practiced in accordance with the Reference Examples given herein below, unless otherwise stated.

Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wtΛvt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.

Reference Example Salts of 2-amino-2-deoxy-D-glucopyranose, glucosamine hydrochloride G4875, glucosamine 2-sulfate G7889, and glucosamine 6- phosphate G5509, were purchased from Sigma Chemical Co., and used in the following Examples.

Example 1 : Effect of 2-amino-2-deoxy-D-glucopyranose on tube formation of HUVEC

To assay tube formation, human umbilical vein endothelial cells (HUVEC) were isolated from freshly obtained cords after cesarean section. Cells were cultured and identified by immunocytochemical staining with anti- Factor VIII antibody. HUVEC grown on Matrigel (BD Bioscience, Bedford, MA, USA), were treated with 1 mg/ml of each salt of 2-amino-2-deoxy-D- glucopyranose, and further incubated at 37 ^°C for 8-16 hrs. As a control, the procedure was repeated without 2-amino-2-deoxy-D-glucopyranose.

Fig. 1A shows the tubular network formed as a result of neovascularization when the cells were grown on Matrigel. However, the microvascular network of HUVEC on Matrigel became disconnected when treated with salts of 2-amino-2-deoxy-D-glucopyranose as shown in Figs. IB, 1C and ID.

Figs. 2A to 2C illustrate the HUVEC grown on Matrigel treated with different concentrations of glucosamine-2-sulfate, and these results show that the formation of microvascular network was inhibited in a dose-dependent manner. The tube areas determined using the image analysis program Image- Pro Plus^® (Media Cybernetics, USA) are summarized in Table 1.

Table 1

As shown in Table 1, the tube area was reduced by 33 and 64% when treated with 0.25 and 2.5 mg/ml of glucosamine, respectively. Example 2: Angiogenesis assay with chorioaliantoic membrane assays (CAM assay)

Fertilized chicken eggs were kept in a humidified incubator at 37^°C. After incubating for three days, 2-3 ml of albumin was removed from each egg with a syringe equipped with a 26-gauge needle. The egg was sealed with a transparent tape and a small window was drilled on the egg. Two days later, an aliquot of 50 μg of glucosamine-2-sulfate was applied to a sterile Thermanox disc (Miles Scientific) and allowed to air dry. The disc was applied to the chorioaliantoic membrane surface through the window and covered with a transparent adhesive tape. The embryo was incubated further for three days at 37 ^°C in a humidified incubator. An appropriate amount of a lipid emulsion was injected into the embryo chorioallantois using a 26-gauge needle so that the vascular network of the chorioaliantoic membrane could be contrasted against the white lipid background. As a control, 15 μl of physiological saline was loaded on a disc instead of 2-amino-2-deoxy-D- glucopyranose following the same procedure. The resulting blood vessels were observed and compared with treated eggs.

In the control group (n=20), capillary vessel formation was not affected in 85% of the embryo (Fig. 3A), while the vessel formation in the disc area (brighter part of the picture) treated with 2-amino-2-deoxy-D-glucopyranose was inhibited in all cases (n= 20, 100%, Fig. 3B).

Example 3 : Animal experiment for angiogenesis (mouse Matrigel model)

The anti-angiogenic activity of 2-amino-2-deoxy-D-glucopyranose was quantified using a mouse Matrigel model.

A 0.4 ml portion of Matrigel containing 50 ng/ml of basic fibroblast growth factor (bFGF) and 50 units/ml of heparin was implanted by subcutaneous injection, and 0.6 mg of 2-amino-2-deoxy-D-glucopyranose per mouse was orally administered twice a day for 4 days. On day 5, the Matrigel was removed and the amount of hemoglobin in the Matrigel was determined. Table 2

As shown in Fig. 4 and Table 2, the 2-amino-2-deoxy-D- glucopyranose-treated group showed a lower level of hemoglobin in

Matrigel, about 34% of that of the control group, demonstrating that 2- amino-2-deoxy-D-glucopyranose has anti-angiogenic activity when it was administered orally.

Example 4 : Acute toxicity of orally administered glucosamine on rat

An acute toxicity test was carried out using SPF SD rats as follows. Aqueous solutions containing various amounts of glucosamine were given to rats via the oral route, and the animals' behaviors were observed. There was no sign of lethality, toxic responses or abnormality at 1.0 g/kg of glucosamine. Biochemical analyses of the animals' blood and urine samples as well as autopsy confirmed that the composition of present invention is safe.

Preparation Example 1 : Preparation of syrup

A syrup containing 2% of glucosamine hydrochloride was prepared using the following ingredients by; dissolving glucosamine hydrochloride, saccharine, and sugar in waπn water, cooling, and adding other ingredients thereto to a volume of 100 ml.

Quantity

Glucosamine hydrochloride 2 g Saccharine 0.8 g

Sugar 25.4 g

Glycerin 8.0 g

Fragrant 0.04 g Ethanol 4.0 g

Sorbic acid 0.4 g

Distilled Water q.s.

Preparation Example 2 : Preparation of tablet

A tablet containing glucosamine-2-sulfate was prepared with the following ingredients by mixing glucosamine-2-sulfate with lactose, com starch and colloidal silica and adding 10%> gelatin solution thereto. Then it was continued by crushing, sieving though a 14 mesh and drying. Finally the remaining ingredients were added thereto and tableting was performed.

Quantity

Glucosamine-2-sulfate 250 g

Lactose 175.9 g

Corn starch 180 g

Colloidal silica 32 g

10% gelatin solution 25 g Com starch 160 g

Talc 50 g

Magnesium Stearate 5 g

Preparation Example 3: Preparation of an injectable solution

Glucosamine-2-sulfate, sodium chloride and ascorbic acid were dissolved in distilled water and sterilized.

Quantity

Glucosamine-2-sulfate 1.0 g

Sodium chloride 0.6 g

Ascorbic acid 0.1 g

Distilled water q.s.

Claims

What is claimed is :

1. A composition for treating or preventing angiogenesis-dependent diseases, which comprises a therapeutically effective amount of 2-amino-2- deoxy-D-glucopyranose or salt thereof.

2. The composition of claim 1, wherein the angiogenesis-dependent diseases is a disease selected from the group consisting of cancer metastasis, angioma, angiofibroma, diabetic retinopathy, premature infant's retinopathy, neovascular glaucoma, comeal disease induced by angiogenesis, involutional macula, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis, acne and arthritis.

3. The composition of claim 1, wherein the salt of glucosamine is glucosamine hydrochloride, glucosamine 2-sulfate, glucosamine pentaacetate or a modified glucosamine.

4. The composition of claim 3, wherein the modified glucosamine is glucosamine 6-phosphate, glucosamine 3-sulfate, glucosamine 6-sulfate, glucosamine 2,3-disulfate, glucosamine 2,6-disulfate, glucosamine 3,6- disulfate, glucosamine 1 -phosphate or glucosamine 6-phosphate.

5. A method for treating or preventing angiogenesis-dependent diseases, which comprises administering 2-amino-2-deoxy-D-glucopyranose or salt thereof to a subject in need thereof in an amount effective for treating or preventing the diseases.